Megakaryocytic Lineage Research Articles

Mesenchymal stem cell infusion for enhancing hematopoietic recovery and addressing cytopenias in CAR-T cell therapy

BackgroundChimeric antigen receptor (CAR)-T therapy has emerged as a promising treatment for hematologic malignancies. However, cytopenia remains one of the most frequent and challenging adverse effects of this therapy.MethodsWe conducted a retrospective analysis of 26 patients with relapsed/refractory aggressive B-cell lymphoma who received CAR-T therapy at our center. Subsequently, to investigate measures to address cytopenias following CAR-T therapy, we isolated and generated murine CAR-T cells and bone marrow-derived mesenchymal stem cells (MSCs), establishing a murine syngeneic CAR-T therapy model. We assessed the impact of MSC infusion on hematopoietic recovery post-CAR-T therapy by evaluating complete blood count, bone marrow hematopoietic stem cells and their subpopulations, bone marrow histomorphology, and hematopoiesis-related genes.ResultsAll patients experienced cytopenias to varying degrees, with complete lineage involvement in half of the patients. Grade ≥ 3 cytopenias were observed in 88.46% of the patients. CAR-T therapy was associated with a higher incidence of biphasic, late-onset, or prolonged cytopenias. Survival analysis indicated that neutropenia and lymphopenia tended to be associated with better prognosis, whereas thrombocytopenia tended to be related to poorer outcomes. Through animal experiments, we discovered that MSCs infusion boosted HSCs and their long-term subpopulations, enhancing hematopoietic recovery, particularly in the megakaryocyte lineage, and mitigating bone marrow damage. Importantly, both in vitro and in vivo experiments demonstrated that MSCs did not compromise the activity or antitumor efficacy of CAR-T cells.ConclusionsOur findings propose MSCs infusion as a promising strategy to address cytopenias, particularly thrombocytopenia, after CAR-T therapy. This approach could help overcome certain limitations of cellular immunotherapy by enhancing hematopoietic recovery without compromising the efficacy of CAR-T cells.HighlightsCytopenia is a frequently observed adverse effect following CAR-T therapy, and it is often characterized by biphasic and prolonged patterns.MSCs play a critical role in promoting hematopoietic recovery and mitigating bone marrow damage in a murine model of CAR-T therapyThe activity and antitumor efficacy of CAR-T cells were not impaired by MSCs.Graphical

Oxazolidinone antibiotics impair ex vivo megakaryocyte differentiation from hematopoietic progenitor cells and their maturation into platelets.

Oxazolidinones (linezolid and tedizolid) adverse reactions include thrombocytopenia, the mechanism of which is still largely unknown. In cultured cells, oxazolidinones impair mitochondrial protein synthesis and oxidative metabolism. As mitochondrial activity is essential for megakaryocyte differentiation and maturation into platelets, we examined whether oxazolidinones impair these processes ex vivo and alter, in parallel, the activity of mitochondrial cytochrome c-oxidase (CYTOX; enzyme partly encoded by the mitochondrial genome) and cell morphology. Human CD34+ cells were isolated, incubated with cytokines (up to 14 days) and clinically relevant oxazolidinone concentrations or in control conditions, and used for (i) clonogenic assays [counting of megakaryocyte (CFU-Mk), granulocyte-monocyte (CFU-GM), burst-forming unit-erythroid (BFU-E) colonies]; (ii) the measure of the expression of megakaryocyte surface antigens (CD34 to CD41 and CD42); (iii) counting of proplatelets; (iv) the measurement of CYTOX activity; and (v) cell morphology (optic and electron microscopy). Oxazolidinones caused a significant decrease in BFU-E but not CFU-Mk or CFU-GM colonies. Yet, the megakaryocytic lineage was markedly affected, with a decreased differentiation of CD34+ into CD41+/CD42+ cells, an abolition of proplatelet formation and striking decrease in the numbers of large polylobulated nucleus megakaryocytes, with a complete loss of intracellular demarcation membrane system, disappearance of mitochondria, and suppression of CYTOX activity. These alterations were more marked in cells incubated with tedizolid than linezolid. These data suggest that oxazolidinones may induce thrombocytopenia by impairing megakaryocytic differentiation through mitochondrial dysfunction. Pharmacological interventions to prevent this toxicity might therefore be difficult as mitochondrial toxicity is most probably inherently linked to their antibacterial activity.

CDK9 phosphorylates RUNX1 to promote megakaryocytic fate in megakaryocytic-erythroid progenitors

AbstractThe specification of megakaryocytic (Mk) or erythroid (E) lineages from primary human megakaryocytic-erythroid progenitors (MEPs) is crucial for hematopoietic homeostasis, yet the underlying mechanisms regulating fate specification remain elusive. In this study, we identify RUNX1 as a key modulator of gene expression during MEP fate specification. Overexpression of RUNX1 in primary human MEPs promotes Mk specification, whereas pan-RUNX inhibition favors E specification. Although total RUNX1 levels do not differ between Mk progenitors (MkPs) and E progenitors (ErPs), there are higher levels of serine-phosphorylated RUNX1 in MkPs than ErPs, and mutant RUNX1 with phosphorylated-serine/threonine mimetic mutations (RUNX1-4D) significantly enhances the functional efficacy of RUNX1. To model the effects of RUNX1 variants, we use human erythroleukemia (HEL) cell lines expressing wild-type (WT), phosphomimetic (RUNX1-4D), and nonphosphorylatable (RUNX1-4A) mutants showing that the 3 forms of RUNX1 differentially regulate expression of 2625 genes. Both WT and RUNX1-4D variants increase expression in 40%, and decrease expression in another 40%, with lesser effects of RUNX1-4A. We find a significant overlap between the upregulated genes in WT and RUNX1-4D–expressing HEL cells and those upregulated in primary human MkPs vs MEPs. Although inhibition of known RUNX1 serine/threonine kinases does not affect phosphoserine RUNX1 levels in primary MEPs, specific inhibition of cyclin dependent kinase 9 (CDK9) in MEPs leads to both decreased RUNX1 phosphorylation and increased E commitment. Collectively, our findings show that serine/threonine phosphorylation of RUNX1 promotes Mk fate specification and introduce a novel kinase for RUNX1 linking the fundamental transcriptional machinery with activation of a cell type–specific transcription factor.

Structural basis of MPL activation by thrombopoietin

AbstractMyeloproliferative leukemia protein (MPL), also known as thrombopoietin (TPO) receptor, is a class I cytokine receptor that is expressed on hematopoietic progenitors, promoting growth and differentiation toward the megakaryocyte lineage and is critical for normal platelet production. Mutations in MPL, TPO, or Janus kinase 2 (JAK2) have been implicated in multiple diseases from congenital thrombocytopenias to myeloproliferative neoplasms. The ligand for MPL, TPO, stimulates platelet production by inducing MPL dimerization and results in an active conformation that allows downstream JAK2/STAT5 signaling. Despite the biological importance of this pathway, the molecular signaling mechanism remained unclear. Here, we present a 3.39-Å cryo-electron microscopy structure of the ectodomain of mouse MPL bound to TPO. The structure revealed both low and high affinity sites between MPL and TPO that contain several pathologic mutations. To better understand TPO-driven MPL signaling, we expanded upon this structure by molecular dynamic (MD) simulations to model the full-length human MPL/TPO complex, and showed that MPL D4-D4 domain interactions are functionally relevant in activity assays. To build on our understanding of downstream activation, we added JAK2 to the MPL/TPO complex by MD simulations. This ternary complex illustrates JAK2 dimerization through the pseudokinase domain, illustrates residues important for MPL interactions, and reveals the constitutive activation mechanism of patient mutant V617F. The model also suggests the mechanism of JAK2 tyrosine kinase domain transphosphorylation. Overall, our studies illuminate TPO/MPL/JAK2 signaling mechanisms and provide additional insight into the nature of receptor signaling, which will further benefit human health.

Lenalidomide and pomalidomide modulate hematopoietic cell expansion and differentiation in the presence of MSC

Cytopenia is a well-documented complication in the treatment of hematological malignancies with lenalidomide and pomalidomide. Although prior studies have highlighted direct effects on hematopoietic cells to explain this adverse effect, the involvement of hematopoietic-supportive stroma remains less understood. This study examined the effects of lenalidomide/pomalidomide on the expansion and differentiation of human CD34+ hematopoietic stem/progenitor cells (HSPCs) in vitro, in co-culture with human bone-marrow mesenchymal stromal/stem cells (MSCs). Our findings indicate that lenalidomide/pomalidomide increases the population of immature CD34+CD38− cells while decreasing the number of mature CD34+CD38+ cells, suggesting a mechanism that inhibits early HSPC maturation. This effect persisted across myeloid, megakaryocytic, and erythroid lineages, with MSCs playing a key role in preserving immature progenitors and inhibiting their differentiation. Furthermore, in myeloid differentiation assays augmented by granulocyte-colony stimulating factor, lenalidomide/pomalidomide not only enhanced the presence of CD34+ cells with mature myeloid markers such as CD11b but also reduced the populations lacking CD34 yet positive for these markers, irrespective of MSC presence. Thus, while MSCs support the presence of these immature cell populations, they simultaneously inhibit their maturation. This finding provides novel mechanistic insights into lenalidomide- and pomalidomide-induced cytopenia, and could guide therapeutic strategies for its mitigation.

Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

The LAT Rheostat as a Regulator of Megakaryocyte Activation.

Specifically positioned negatively charged residues within the cytoplasmic domain of the adaptor protein, linker for the activation of T cells (LAT), have been shown to be important for efficient phosphorylation of tyrosine residues that function to recruit cytosolic proteins downstream of immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling. LAT tyrosine 132-the binding site for PLC-γ2-is a notable exception, preceded instead by a glycine, making it a relatively poor substrate for phosphorylation. Mutating Gly131 to an acidic residue has been shown in T cells to enhance ITAM-linked receptor-mediated signaling. Whether this is generally true in other cell types is not known. To examine whether LAT Gly131 restricts ITAM signaling in cells of the megakaryocyte lineage, we introduced an aspartic acid at this position in human induced pluripotent stem cells (iPSCs), differentiated them into megakaryocytes, and examined its functional consequences. iPSCs expressing G131D LAT differentiated and matured into megakaryocytes normally, but exhibited markedly enhanced reactivity to glycoprotein VI (GPVI)-agonist stimulation. The rate and extent of LAT Tyr132 and PLC-γ2 phosphorylation, and proplatelet formation on GPVI-reactive substrates, were also enhanced. These data demonstrate that a glycine residue at the -1 position of LAT Tyr132 functions as a kinetic bottleneck to restrain Tyr132 phosphorylation and signaling downstream of ITAM receptor engagement in the megakaryocyte lineage. These findings may have translational applications in the burgeoning field of in vitro platelet bioengineering.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

Transient Myeloproliferative Disorder in a Neonate without Down Syndrome—A Rare Case Report and Review of the Literature

AbstractTransient myeloproliferative disorder (TMD) is a self-limiting disorder characteristically seen in neonates with Down syndrome with or without somatic mosaicism. Trisomy-21 limited to the hematopoietic lineage alone has been described; awareness of which is very important for appropriate evaluation and counseling in phenotypically normal children.We report a newborn with TMD who presented at birth with intracranial bleed secondary to thrombocytopenia. Peripheral smear showed 10% blasts and flow cytometry further revealed myeloid blasts of megakaryocytic lineage. The child had no phenotypic features of Down syndrome.Cytogenetic analysis (fluorescence in situ hybridization) and the conventional karyotyping from peripheral blood showed trisomy-21 in blast cells and the findings completely cleared with peripheral clearance of blasts. The possibility of Down syndrome with mosaicism was considered, however, repeat conventional karyotyping from peripheral blood at D36 and D60 of life was normal, suggesting the gain of chromosome 21 was restricted to the TMD clones.The child was supported with irradiated platelet transfusions and adequate hydration. Spontaneous resolution with resolution of cytopenias and peripheral clearance of blasts were noted from D10 of life. The child is neurologically normal and growing well.Very few reports of TMD in newborn babies without Down syndrome have been described in the literature. Awareness about the diagnostic entity of TMD even without Down syndrome would help in appropriate management and counseling.

HMGA2 promotes platelet-neutrophil complex formation and pulmonary tissue damage in myelodysplastic syndromes

AbstractHigh mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that functions as an oncogene in various cancers. Although overexpression of HMGA2 has been reported in several myeloid malignancies, its role varies considerably in different disease contexts. Here, we identified a distinct role of HMGA2 as a mediator of noninfectious pneumonia in myelodysplastic syndrome (MDS). The expression level of HMGA2 in CD34+ hematopoietic stem cells and progenitors (HSC/Ps) was significantly associated with the incidence of noninfectious pneumonia, a common systemic complication in patients with MDS. Consistent with this clinical investigation, HMGA2 overexpression in a mouse model of an MDS-associated mutation led to the development of lethal noninfectious pneumonia. Mechanistically, HMGA2 overexpression conferred a megakaryocytic lineage bias to HSC/Ps and contributed to platelet activation in MDS mice. P-selectin–positive activated platelets interacted with MDS clone–derived neutrophils that exhibit increased susceptibility to cell death and formed platelet-neutrophil complexes (PNCs). Both the frequency of PNCs and neutrophil cell death within the lung microenvironment increased in MDS mice overexpressing HMGA2. Genetic inhibition of P-selectin attenuated pulmonary tissue damage in MDS mice. These findings indicate that PNCs could be a new therapeutic target for noninfectious pneumonia in patients with MDS and provide new insights into the mechanistic basis of the systemic complications of MDS.

An intricate regulatory circuit between FLI1 and GATA1/GATA2/LDB1/ERG dictates erythroid vs. megakaryocytic differentiation.

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP‑like cell line HEL western blotting, RT‑qPCR, lentivirus‑mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation‑specific (ETS) transcription factor friend leukemia integration factor 1 (Fli‑1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1‑mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation‑specific‑related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA‑mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.

Abstract 7186: In vitro evaluation of myelosuppressive effects of ATRi tuvusertib and ATMi lartesertib (M4076), alone and in combination

Abstract Introduction: In cancer therapy, combinations of drugs interfering with different molecular pathways enhance efficacy and reduce resistance development, but may also increase the risk of toxicity. DNA damage repair inhibitors (DDRi) induce on- and off-target adverse events including hematological and gastro-intestinal toxicities1, which impact the therapeutic window. We evaluated the hematopoietic effect of ATRi tuvusertib and ATMi lartesertib, alone and in combination, on erythroid, megakaryocytic and myeloid bone marrow cell lineages. Methods: Mobilized peripheral blood CD34+ cells were plated in 96-well plates in specific media cultures allowing growth and differentiation of all three lineages. The cells were cultured for a total of 7 (neutrophils and platelets) to 10 (erythroids) days in the absence of drugs to generate maturing populations. Various concentrations of tuvusertib and lartesertib were incubated in cell media and assessed using LC/MS methods to ensure they remained chemically stable for the duration of the experiments. After in vitro differentiation, the lineages were cultured in the continuous presence of various concentrations of tuvusertib (0.003 to 3 µM) and/or lartesertib (0.01 to 10 µM) encompassing an unbound plasma concentration range evaluated in phase 1 studies of these agents and their combination. Cells were evaluated in triplicate by flow cytometry on day 3 and 7 of drug exposure for neutrophils (CD34, CD38, CD33, CD15), megakaryocytes and platelets (CD41, CD42b), and erythroid cells (CD71, CD235a). In another set of experiments, to simulate a "drug holiday", cells were exposed to tuvusertib, lartesertib and their combination for 24 h, washed out and further cultured as described above before evaluation. Results: Tuvusertib and lartesertib were stable in the specific media cultures up to day 7 of drug exposure. Continuous treatment with tuvusertib as a single agent inhibited the hematopoietic cells of all lineages more profoundly than lartesertib as monotherapy. Effects were consistently more pronounced after 7 days than 3 days of drug exposure. Continuous treatment of a combination of tuvusertib and lartesertib inhibited hematopoietic cells of all lineages. Treatment for 24 h followed by wash-out demonstrated a lesser effect compared to continuous treatment. Conclusion: The phenotypic and functional changes induced by tuvusertib and lartesertib on in vitro cultures of erythroid, myeloid, and megakaryocytic cells are consistent with clinical observations in monotherapy studies, suggesting these models may be used to better understand and predict the hematological effects of monotherapies and combinations in the clinic. In vitro washout experiments may help support the concept of intermittent clinical regimens and warrant further investigation. 1. Martorana, et al. Cancers (Basel) 2022;14(4):953 Citation Format: Jatinder Kaur Mukker, Rohan Kulkarni, Yongzhao Huang, Emer Clarke, Ioannis Gounaris, Anup Zutshi, Karthik Venkatakrishnan, Anna Tafuri. In vitro evaluation of myelosuppressive effects of ATRi tuvusertib and ATMi lartesertib (M4076), alone and in combination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7186.

Aryl hydrocarbon receptor activation alters immune cell populations in the lung and bone marrow during coronavirus infection.

Respiratory viral infections are one of the major causes of illness and death worldwide. Symptoms associated with respiratory infections can range from mild to severe, and there is limited understanding of why there is large variation in severity. Environmental exposures are a potential causative factor. The aryl hydrocarbon receptor (AHR) is an environment-sensing molecule expressed in all immune cells. Although there is considerable evidence that AHR signaling influences immune responses to other immune challenges, including respiratory pathogens, less is known about the impact of AHR signaling on immune responses during coronavirus (CoV) infection. In this study, we report that AHR activation significantly altered immune cells in the lungs and bone marrow of mice infected with a mouse CoV. AHR activation transiently reduced the frequency of multiple cells in the mononuclear phagocyte system, including monocytes, interstitial macrophages, and dendritic cells in the lung. In the bone marrow, AHR activation altered myelopoiesis, as evidenced by a reduction in granulocyte-monocyte progenitor cells and an increased frequency of myeloid-biased progenitor cells. Moreover, AHR activation significantly affected multiple stages of the megakaryocyte lineage. Overall, these findings indicate that AHR activation modulates multiple aspects of the immune response to a CoV infection. Given the significant burden of respiratory viruses on human health, understanding how environmental exposures shape immune responses to infection advances our knowledge of factors that contribute to variability in disease severity and provides insight into novel approaches to prevent or treat disease.NEW & NOTEWORTHY Our study reveals a multifaceted role for aryl hydrocarbon receptor (AHR) signaling in the immune response to coronavirus (CoV) infection. Sustained AHR activation during in vivo mouse CoV infection altered the frequency of mature immune cells in the lung and modulated emergency hematopoiesis, specifically myelopoiesis and megakaryopoiesis, in bone marrow. This provides new insight into immunoregulation by the AHR and extends our understanding of how environmental exposures can impact host responses to respiratory viral infections.

Myosin light chain 6 (Myl6) interacts with kindlin-3 and is required to support integrin αIIbβ3 activation in platelets in mice

BackgroundKindlin-3 in platelets plays an essential role in supporting integrin αIIbβ3 activation, platelet spreading, aggregation, and clot retraction by binding to the integrin β3 cytoplasmic tail. However, the mechanism by which kindlin-3 mediates the crosstalk between integrin αIIbβ3 and myosin in platelets remains unknown. ObjectivesTo examine the role of myosin light chain 6 (Myl6) in supporting integrin αIIbβ3 activation in platelets. MethodsMyl6fl/flPF4-Cre mice with a deficiency of Myl6 in the megakaryocyte lineage were generated, and integrin αIIbβ3 activation in Myl6-deficient platelets was analyzed. ResultsWe identified a novel kindlin-3 binding protein, Myl6, an essential light chain of myosin in platelets. Myl6fl/flPF4-Cre mice exhibited significant macrothrombocytopenia resulting from defective proplatelet formation. In the absence of Myl6, integrin αIIbβ3 activation in platelets was significantly suppressed, and platelet aggregation was substantially impaired. Interestingly, the deficiency of Myl6 in platelets preferentially affected the binding of a multivalent ligand compared to a monovalent ligand to integrin αIIbβ3 upon activation, indicating that Myl6 may contribute to the avidity modulation of integrin αIIbβ3 by binding to kindlin-3. Furthermore, blood coagulation ability was impaired in Myl6fl/flPF4-Cre mice, and consistently, these mice exhibited defects in both hemostatic and thrombotic functions. ConclusionIn summary, these results suggest that Myl6, as a novel kindlin-3 binding partner, is required to support integrin αIIbβ3 activation in platelets, which plays an important role in both hemostasis and thrombosis.

Unrevealing Hematopoietic Reconstruction Discrepancies after Allo-UCBT and Allo-PBSCT at Single Cell Resolution

Umbilical cord blood (UCB) and G-CSF mobilizing peripheral blood stem cell (G-PBSC) are the main sources of hematopoietic stem cells for transplantation on clinic. Clinical data have shown that hematopoietic reconstruction in allogeneic cord blood transplantation (allo-UCBT) is significantly delayed compared with allogeneic peripheral blood stem cell transplantation (allo-PBSCT), suggesting that the cellular composition and reconstruction dynamics of UCB are significantly different from that of G-PBSC. Following on the heels of our recently study that found HSC/MPPs show differentiation bias to megakaryocytic-erythroid lineage and strong proliferation tendency, and defined a neutrophil progenitor population named S100A hi Neu2 which is inversely associated with the incidence of GVHD after allo-PBSCT (Yingying Huo et al, Science Immunology 2023), we next aimed to analyze the differences of hematopoietic reconstitution between allo-UCBT and allo-PBSCT and the causes of delayed hematopoietic reconstitution after allo-UCBT. In this study, we collected peripheral blood (PB) and bone marrow (BM) samples from 8 patients with bone marrow failure syndrome (BMFs) who received allo-UCBT (n=4) or allo-PBSCT (n=4) at different time points (d14, d21, d30, d60, d90, d180, d365) after transplantation. At the same time, UCB, G-PBSC and BM of normal children were collected as controls. Lin -CD34 + cells from BM, G-PBSC and UCB were separated by FACS for STRT-seq, the total nucleated cells (TNCs) of all samples were isolated for 10X transcriptome sequencing. We used 276628 hematopoietic cells to map the single cell transcriptome profiles of hematopoietic reconstruction by different transplantation methods and annotated 44 cell types. Compared with the G-PBSC, the immunogenicity of HSC/MPPs in Lin -CD34 + cells derived from UCB decreased, the proportion increased, and the differentiation bias of HSC/MPPs to monocytic and erythroid lineage was stronger, but the differentiation ability to myeloid and megakaryocytic lineage was decreased, the proportion of EBM, MEP and CMP decreased. For TNCs, the proportion of T cells and NK cells increased, and NK_CD56dim_CD16 had a stronger immune function. Compared with allo-PBSCT, the differentiation of HSC/MPPs and CMP into neutrophils in the early stage after allo-UCBT was weakened. The proportion of MEP cells decreased, and the differentiation ability of HSC/MPPs to MEP, HSC/MPPs and MEP to Mk was weakened. In summary, we found that the delayed reconstruction of neutrophils and platelets in patients with allo-UCBT are related to the nature of the graft, suggesting the importance of reasonable intervention of HSC derived from UCB to accelerate platelet implantation.

Structural Basis for Activation of the Thrombopoietin Receptor (MPL) By Its Ligand (TPO)

Myeloproliferative leukemia protein (MPL), also known as thrombopoietin receptor, is a class I cytokine receptor expressed on hematopoietic progenitors that is critical for normal platelet production by promoting myeloid progenitor growth and differentiation toward the megakaryocyte lineage. The ligand for MPL, thrombopoietin (TPO), stimulates platelet production through binding MPL via two sequential sites, a high- and then a low-affinity interface that together induce MPL dimerization and result in an active conformation allowing downstream JAK2/STAT5 signaling. Despite intense interest in characterizing the molecular mechanism of the interaction between MPL and TPO over the last several decades, no experimental structural evidence has been reported. Here we present a 3.39 Å cryo-EM structure of the ectodomain of mouse MPL bound to TPO. TPO features both low and high affinity sites that each bind to one MPL monomer in agreement with previous functional studies. Our structure reveals that several patient mutations associated with congenital amegakaryocytic thrombocytopenia (CAMT) and aplastic anemia are found at MPL-TPO interaction sites. The structure further explains additional patient mutations, as well as published experimental mutations that have previously been the basis of structure-function studies. Building on this structure, we generated a molecular dynamics simulation model of the full length human MPL-TPO complex, revealing that TPO binding to full length MPL results in unexpected MPL-MPL intramolecular interactions that stabilize the dimer to allow for efficient signaling. Functional characterization of deleterious mutations predicted by our structure and modelling have revealed additional residues critical to MPL-TPO and MPL-MPL interactions, providing further validation of our proposed structure. Overall, our studies reveal new insight into a receptor-cytokine interaction critical for normal hematopoiesis and platelet production that are frequently mutated in disease, provide the structural basis underlying existing experimental findings, and provide additional insight into the nature of hematopoietic growth factor signaling which could benefit future therapeutic research.

Erythroid Progenitor Cells in the Murine Bone Marrow: Parallels with Human Counterparts and Response to Acute Anemia

In mammals, the erythron consists of lineage-specific progenitor cells that transition to morphologically identifiable precursors that ultimately enucleate to become mature red blood cells. Early- and late-stage erythroid progenitors, BFU-E and CFU-E respectively, were originally defined by their capacity to generate colonies of erythroid cells in semisolid media. We have refined a flow cytometry-based strategy based on Pronk, et al. (2007) and Tusi, et al. (2018) that parallels similar strategies developed for human erythroid progenitor cells (Yan et al., 2021) to facilitate identification and isolation of progressive stages of primary murine erythroid progenitors, termed EP1 to EP4, in the bone marrow. Sorted erythroid progenitor (EP1 to EP4) populations were cultured in colony-forming assays. EP1 consists almost entirely of BFU-E, while EP4 was highly enriched in CFU-E, and the intermediate EP2 and EP3 populations progress from immature to more mature erythroid colony-forming progenitors. Global RNA-Seq studies were performed on three replicates of primary bone marrow EP1-4 from 9-12 week old outbred mice. EP1/2 and EP3/4 each shared the most similarities, consistent with the enrichment of BFU-E in the former and CFU-E in the latter. We also analyzed the global transcriptome of EP1-4 derived from in vitro culture of human CD34+ hematopoietic stem and progenitor cells to compare with the murine counterparts. Approximately two-thirds of differentially expressed genes displayed similar patterns as murine and human EP1 transition progressively to EP4. The largest transition in gene expression occurred between EP2 and EP3. Analysis of genes differentially expressed between EP2 and EP3 both in human and in mouse (adj. p-value <0.05, 361 down and 242 up) revealed marked downregulation of transcription factors expected from previous studies of erythroid progenitors (Gata2, Cd34), as well as those associated with multipotential, myeloid, and megakaryocyte lineages (Sp1 and Runx1). Upregulated transcription factors included prototypical factors associated with erythropoiesis (Klf1, Gata1 and Tal1), as well as Srebf1, a central regulator of cholesterol homeostasis. In both species the transition from EP2 to EP3 was characterized by downregulation of the gene ontology terms associated with myeloid and megakaryocyte development, and the upregulation of gene ontology terms associated with erythrocyte development, cell cycle, heme metabolism, and VLDL particle assembly. Interestingly, cholesterol content increases as EP1/2 transition to EP3/4 in both primary murine and human bone marrow. These data indicate a shared progression of erythroid progenitors can be proactively delineated in human and mouse systems. To better define the response of erythroid progenitors to stress, we established an acute anemia model in mice, reproducibly lowering the hematocrit by half via phlebotomy. Induction of acute anemia leads to a marked expansion of EP3 and EP4 at 48 hours both in the bone marrow and in the spleen. Additionally, these expanded EP3/4 populations were characterized by an increase percentage of the cells in S-phase. Principal component analysis of global transcriptomic studies of EP1-4 isolated from the marrow 48 hours after the induction of anemia revealed parallel differentiation programs when compared to their steady-state murine counterparts. 70% of the shared genes upregulated in the transition from EP2 to EP3 were upregulated to a greater extent in response to anemia. Upregulated genes, particularly in EP3/4, included known EPO-responsive genes (Pim1, Socs2, Tfrc, and Cited4), as well as hypoxia-associated genes. Interestingly, key cholesterol biosynthesis genes (Hmgcr, Hmgcs1, and Sqle) were also further upregulated in anemic versus steady-state EP3/4. This latter finding suggests that cholesterol homeostasis may play a key role in the erythroid progenitor response to stress. We conclude that late-stage erythroid progenitor (EP3/4) expansion is a specific and integral component of regenerative erythropoiesis. The identification and isolation of stage-specific murine erythroid progenitor cells that are highly analogous to their human counterparts will facilitate an improved understanding of normal and disordered erythropoiesis.

Thrombocytosis and Megakaryocyte Changes Associated with Pure Red Cell Aplasia

J.A., R.G.W, and G.R. contributed equally to this abstract. Introduction: Acquired pure red cell aplasia (PRCA) is a rare hematological disorder characterized by normocytic, normochromic anemia with severe reticulocytopenia and a notable absence of identifiable erythroid precursors in the bone marrow. PRCA is often without a clear etiology and can be associated with conditions such as Large Granular Lymphocytic (LGL) Leukemias, B-cell dyscrasias, thymomas, immunodeficiencies or infections. Discovery of an IgG inhibitor in primary autoimmune pure red cell aplasia (PRCA) has been an immensely significant breakthrough. This finding has not only shed light on the critical role of IgG inhibitors in the pathophysiology of PRCA but has also underscored the importance of aberrant T cell activity in this condition. Thrombocytosis refers to an elevation in platelet count of >450,000/μL, which is not a typical feature associated with PRCA. Based on observations in our clinical practice of patients with PRCA with concomitant thrombocytosis, we screened our cohort for the combination of these features to discover potential mechanisms underlying this phenomenon. Methods: In order to address this objective, we conducted a retrospective analysis of medical records from 90 patients diagnosed with acquired PRCA at the University of Texas Southwestern and Cleveland Clinic Foundation between 2000 and 2022. Our primary goal was to determine the frequency of thrombocytosis among these patients. For those in the subset displaying thrombocytosis, longitudinal hemoglobin and platelet counts before and after treatment of the condition were also collected for relevant comparisons utilizing paired t-tests after assessing for normality with Shapiro-Wilk test. Results: Among a cohort of 90 patients diagnosed with acquired PRCA, 12 individuals exhibited thrombocytosis at the time of PRCA diagnosis (Table 1). Demographically, this subset of individuals had a median age of 53.5, and a 3:1 ratio of women to men. 66.7% of those with thrombocytosis also exhibited megakaryocyte hyperplasia on bone marrow biopsy. Molecular analysis ruled out the presence of myeloproliferative neoplasms, and blood work was obtained ruling out iron deficiency. Patients were treated for PRCA with a variety of medications including prednisone, cyclosporine, tacrolimus, rituximab, and cyclophosphamide. After treatment, 8 of the 12 patients demonstrated complete resolution of their PRCA, defined as transfusion independence with hemoglobin levels above 10. Further examination of those 8 patients with resolution of PRCA revealed statistically significant differences in mean hemoglobin and platelet counts before and after treatment (Figure 1). Specifically, the mean hemoglobin count increased from 7.9 (SD 2.5) g/dL to11.6 (1.8) g/dL (p=0.0168), while the platelet count decreased from 557,300 (SD 104,700) /μL to 414,400 (SD 109,600) /μL (p=0.0049). Conclusion: Our report is the most comprehensive case series to date reporting on thrombocytosis in patients with acquired PRCA. We speculate that the arrest of erythropoiesis generating PRCA may lead to skewing of precursors towards the megakaryocytic lineage. Alternatively, EPO production secondary to PRCA could stimulate TPO receptors and thus stimulate platelet production. Clinicians should be aware of this particular PRCA presentation. Further investigations will aid in elucidating whether one of these mechanisms may also be playing a role in PRCA-related thrombocytosis. The significance of thrombocytosis or megakaryocyte alterations in PRCA remains enigmatic, necessitating further investigations to elucidate their role in disease pathophysiology, as well as their utility as biomarkers for treatment response prediction. The limitations of this study encompass a small sample size and the absence of post-treatment bone marrow biopsy results, which would have facilitated the visual assessment of any resolved megakaryocyte abnormalities. Future directions include examining megakaryocyte hyperplasia and other megakaryocyte abnormalities such as hyper- and hypo-lobation associated with PRCA.

Epitope Edited Hematopoietic Stem Cells Allow Immune-Based In Vivo Selection of Genome-Engineered Cells

Hematopoietic stem/progenitor cell (HSPC) transplantation (HSCT) offers curative options for patients affected by conditions for which the substitution of endogenous hematopoiesis with genetically corrected or healthy donor-derived cells can halt the pathogenic process. Nonetheless, the short and long-term adverse effects of genotoxic conditioning remain a substantial barrier to a wider application of HSCT and gene therapy. HSCT survivors suffer from life-long irreversible consequences, such as chronic graft-versus-host-disease (GvHD), infertility, susceptibility to viral infections, organ damage (including renal, liver and lung) and increased risk of secondary malignancies. Whereas monoclonal and toxin-conjugated antibodies (Abs) targeting HSPCs have been proposed as an alternative to chemo/radiotherapy, the mechanism of action and pharmacokinetics of these agents hamper their safe clinical use due to the risk of on target killing of transplanted HSPCs. To avoid this, the cell product is not administered at peak myeloablation to allow for washout of the conditioning agent, further impairing engraftment and therapeutic potential. Here, we show that precise editing of the targeted epitope in HSPCs can endow hematopoietic lineages with selective resistance to monoclonal Abs (mAbs) without affecting stem cell function. This strategy allows improved myeloablation - which can continue after HSCT - and progressive enrichment of gene-modified cells. We have identified amino-acid changes in the extracellular domain of KIT (CD117) that abrogate the binding of therapeutic mAbs without affecting surface expression, ligand affinity, kinase phosphorylation, downstream signaling, transcriptional and proliferative response (Fig.A). We exploited adenine base editing (BE) to introduce these mutations with high efficiency (~80%) in CD34+ HSPCs, with preservation of long-term repopulating and multilineage differentiation capacity in primary and secondary recipient mice. To ensure full preservation of KIT functionality, we performed growth analysis, RNA sequencing, and phospho-proteomic analyses of epitope-edited HSPCs stimulated with KIT ligand (stem cell factor, SCF) and observed no difference in the proliferation, transcriptional, and phosphorylation response compared to controls. In vitro differentiation of edited HSPCs toward myeloid, macrophage, classical dendritic, granulocytic and megakaryocytic lineages was similar to controls and did not result in counterselection of edited cells. Lineage-differentiated cells showed preserved functionality, including: reactive oxygen species production by myeloid cells, E.coli phagocytosis by macrophages, M1/M2-like macrophage polarization, phospho-flow profiling of IL4-, PMA/ionomycin-, GM-CSF-, IFN type-I-, IL-6- and LPS-stimulated myeloid cells, HLA class-II/CD86 upregulation by DCs, induction of granulocyte NETosis and generation of hyperdiploid megakaryocytes. By plating edited HSPCs with increasing concentrations of the mAb, we observed preserved expansion of edited HSPCs in response to SCF while control cells were inhibited in a dose-dependent manner. To improve HSPCs therapies for hemoglobinopathies, such as sickle cell disease, we multiplexed BE of the KIT epitope and two BCL11A erythroid enhancer motifs. We obtained editing efficiencies comparable to single editing and the erythroid progeny of triple-edited cells showed upregulation of HbF, as expected (~40%). In a competitive xeno-transplantation setting, with co-injection of multiplex BE and AAVS1-BE control CD34+ cells, mice treated with KIT mAb showed a 2-fold co-enrichment of both KIT and BCL11A editing when compared to mock treated controls, confirming the possibility to select gene-modified cells up to therapeutic thresholds. Similarly, mAb conditioning combined with epitope-edited grafts allowed human hematopoietic substitution in mice pre-humanized with unedited CD34+ HSPCs (89% substitution with 7.3-fold higher absolute cell counts, Fig.B), compared to AAVS1-edited controls that failed to engraft. We envision a new paradigm for non-genotoxic conditioning based on transplantation of epitope engineered HSPCs resistant to immunotherapeutic agents to achieve engraftment and in vivo selection of gene corrected HSPC with substantial advantages over alternatives in terms of safety and tolerability.

Gene Therapy Expressing Platelet-Derived Factor VIII for Correction of Hemophilia Α with a History of Inhibitors

Introduction: Platelet targeted gene therapy with human factor VIII has established hemostasis in murine and canine models with severe hemophilia A withouteliciting inhibitory antibodies. Here-in we describe feasibility, safety and efficacy of the first subject treated on a first-in-human phase 1 trial that targets factor FVIII synthesis and storage within platelet α-granules for delivery at the site of vascular injury for hemophilia A (NCT03818763). Method: Eligible are severe hemophilia A patients ≥18 years with a history of inhibitors to factor VIII (≥0.6 Bethesda Units [BU]/ml). Following mobilization, autologous CD 34+ cells (≥6 x 10 6/kg) are collected and transduced with a lentiviral vector encoding the ITGA2B gene promoter for ectopic expression of human B-domain-deleted factor VIII within the megakaryocyte lineage. The transduced cells are infused after reduced intensity cytoreduction with fludarabine (120 mg/m 2) and melphalan (120 mg/m 2) followed by a washout period of ≥24 hours. The primary endpoints of feasibility and safety are defined as: 1) feasibility of the cell manufacturing procedure by the availability of ≥4 x 10 6/kg transduced clinical grade CD34+cells, cell viability ≥70% and undetectable microbial contamination and 2) safety defined as hematopoietic recovery ≤28 days of infusion and absence of ≥ grade 3 toxicity (CTCAE version 5.0). Outcome: Subject 1 is a 29-year-old male with severe hemophilia A who developed inhibitors to factor VIII (2.6 BU/ml) in his first year of life. Immune tolerance to factor VIII had been established with a non-detectable inhibitor titer at enrollment. Hemostatic prophylaxis was maintained with emicizumab weekly and recombinant factor VIII for breakthrough bleeding. His annualized bleeding rate was 16 in the 12-month period preceding infusion. The cell product was the result of 1 day of collection yielding 6.73 x 10 6 /kg total viable CD34+ cells post-transduction and vector copy number by qPCR of 1.16 copies/cell. Megakaryocyte factor VIII:C levels were 0.00 mU/10 6 before transduction and 101.32 mU/10 6 after transduction. The cell product satisfied release criteria. Neutrophil recovery (≥0.5 x 10 9/L) and platelet transfusion independence (≥50 x 10 9/L) was achieved 15 days post-infusion and sustained for >12 months. The duration of hospitalization was 21 days with no re-admissions. There was no breakthrough bleeding during collection and hospitalization. During and post-infusion there was no unexpected toxicity ≥ grade 1. He has not required immune suppression. Emicizumab was discontinued 3.6 months post-infusion after demonstration of whole blood vector copy number at 1 and 3-months. There has been no spontaneous bleeding or a need for “on demand” factor VIII after discontinuing emicizumab. Secondary outcomes are summarized in Table 1. No replication competent lentivirus was detected through month 12 post-infusion. Integration site analysis was carried out, and none of the samples tested through 12 months contained cell clones which exceeded 20% relative abundance. Transduced cells were highly polyclonal. Integration site analysis showed no enrichment of integration near cancer-associated genes in the cell product nor in whole blood cell lineages post-infusion. Conclusion: We report feasibility, safety and efficacy in the first subject with severe hemophilia A and a history of inhibitors who received lentiviral vector gene therapy directed to induce megakaryocytes to synthesize and store factor VIII within platelets. These findings extend the potential to treat severe hemophilia A patients who are not eligible for valoctcogene roxaoarvovec (AAV5-hFVIII-SQ).